Figure 1.
Egg deposition is more abundant within PP-associated vasculature.
(A) Outline of experimental S. mansoni infection. (B) Numbers of eggs/g intestinal segments containing PP (+PP), or adjacent segments lacking PP (−PP) at 6, 8, and 12 weeks post-infection (pi). Data for −PP and +PP gut samples from the same mouse shown by connecting lines, n = 10–12, *P<0.05 or ***P<0.001, paired T test. (C) Angiograms of +PP vasculature (rhodamine dextran; red) in naïve mice, or 8 and 12 weeks pi. Dotted line demarks PP; arrows indicate schistosome eggs (auto-fluorescence). (D) Ratio of vascular area in PP+/PP− gut (imaged at the anti-mesenteric serosal face or 90° lateral to the anti-mesenteric face) based on angiograms in naïve mice or 8 and 12 weeks pi; min 3 PP/mouse. (E) 3D confocal images of PP stained for B cell follicles (green B220+) and HEV (red MAdCAM+); Arrows = schistosome eggs (auto-fluorescence). (F & G) Quantification of HEV from 3D rendered confocal images of gut sections stained for MAdCAM+ (red), ***P<0.001, *P<0.05 cf. naïve, 1-way ANOVA with Tukey's post-hoc. Data is mean (+SEM), n = 3–4 mice/group and is representative of 2–3 independent experiments.
Figure 2.
Egg deposition within PP disrupts lymphoid microarchitecture and causes loss of cellularity.
(A) DsRed and eYFP expression demarcate B cell follicles and inter-follicular T cell zones of PP. Images are 3D rendered multi-photon confocal display of excised PP tissue derived from VaDsRed/CD19+ eYFP double fluorescent reporter mice (red = CD3+ cells; green = CD19+ cells; blue = second-harmonic generation by collagen fibres). Auto-fluorescent egg (arrows) proximal to PP at 6 and 8 weeks pi. All data are representative of two independent experiments, n = 3 mice. (B) Stereo fluorescent stereomicroscope images of PP in naïve, and infected CD3+/CD19+ reporter mice. Auto-fluorescent eggs (arrows) co-localise to T and B cell zones of PP at 14 weeks (lower panel). (C) 3D multiphoton confocal image of PP egg-granuloma (enlargement of section inset shown in B as dotted line); Sm = autofluorescent egg; arrows = cellular infiltrate; blue = second-harmonic generation by collagen fibres. (D) Numbers of CD11b+ cells, CD11b+SigLecF-F4/80+ macrophages, and CD11b+SigLecF+F4/80lo eosinophils in PP cell suspensions enumerated by flow cytometry in naïve mice, or after infection. (E) CD19+ PP follicle area in double reporter mice (min 3 PP/mouse). (F) Total viable PP, B220+ and IgA+B220+ B cell yields/mouse and (G) proportion of B220+ cells of total PP. (H) Stereo fluorescent stereomicroscope images of mLN in situ from naïve and infected CD3+ (red)/CD19+ (green) reporter mice. (I) mLN follicle area, (J) total viable cell and B220+ B cell yields in the mLN of naïve and infected CD3+T cell/CD19+B cell double reporter mice. All data; *P<0.05, **P<0.01, ***P<0.001, cf. naïve, 1-way ANOVA with Tukey's post-hoc. Bars are means (+SEM), n = 3–5, representative of 2–3 independent experiments.
Figure 3.
Egg deposition and their secretions elicit damage to PP stroma.
(A) Viable (annexinV−/propidium iodide−) CD45− but not CD45+ cells decline in PP cell suspensions after infection; *P<0.05, ***P<0.001, cf. naïve, 1-way ANOVA with Tukey's post-hoc. (B) Upper panels: 3D confocal imaging of PP showing B220+ cell follicles (green, yellow outline), desmin+/smooth muscle actin− fibroblast-like reticular cells (FRC, blue) and desmin+/smooth muscle actin+ blood vessel basement membranes (pink). Dashed lines demarcate void space in FRC tissue. Middle panels: high magnification of desmin+ FRC within PP interfollicular region. Dashed lines demarcate void space in FRC tissue. Lower panels: 3D confocal imaging of PP showing B220+ cell follicles (yellow outline), CR2/1hi follicular dendritic cells (FDC, white) and MAdCAM+ high endothelial venules (red). Intravascular S. mansoni eggs (red autofluoresence) are indicated by arrows. (C) FRC+ and FDC+ tissues as a proportion of inter-follicular volume, or follicular volume: min 3 PP/mouse, *P<0.05, cf. naïve, unpaired T-test. Representative of 2–3 independent experiments, n = 4–5. (D) Phase-contrast images of immature or mature eggs co-cultured with OP9 bone marrow fibroblast stroma (bars = 100 µm). (E) Metabolic activity of fibroblasts 72 h after co-culture as determined by Alamar Blue. (F) Phase contrast images of L929 fibroblasts 72 h post culture in the presence, or absence, of 10 µg/ml secretions from mature eggs (ESP). (G) Metabolic activity determined by Alamar Blue reduction of L929 fibroblasts co-cultured for 72 hr with different concentrations of ESP; untreated control = dashed line. *P<0.05, ***P<0.001, cf. naïve or untreated control, 1-way ANOVA with Tukey's post-hoc. Data are means (+SEM), n = 3–5, representative of 3 independent experiments.
Figure 4.
PP are required to sustain optimum egg transmission and limit host morbidity.
(A) Quantitative analysis of adult schistosome worm recoveries following perfusion of the hepatic portal system in WT or PPnull mice. (B) Cytokine secretion by mLN cells from WT and PPnull mice 8 weeks post-infection following in vitro stimulation with anti-CD3 mAb, or soluble egg antigen. (C) Masson's Trichrome stained histological sections of duodenum (blue = collagen) at post-infection in WT or PPnull mice (red dotted lines = extent of egg granulomas). (D) Reduction in granuloma areas at 16 cf. 8 weeks; min 4 granulomas/mouse, **P<0.01, ***P<0.001, unpaired T test: nd = no difference between WT and PPnull. (E) Numbers of faecal eggs from WT and PPnull mice 8–16 weeks pi, and (F) tissue eggs in the small intestine and liver at week 16; both normalised to female worm burden. (G) Levels of AST enzyme in the serum of naïve or infected mice. (H) Incidence of morbidity of ‘moderate severity’; P = 0.0507, survival analysis. Statistical analysis is for PPnull versus WT mice, *P<0.05, **P<0.01, ***P<0.001, unpaired T test of log10-transformed data (E) or raw data (D, F, G & H). Data is mean+SEM of two independent experiments, (A, B & D; n = 4), or combined data of two independent experiments (E–H; n = 7–13).